Porcine reproductive and respiratory syndrome virus resistant animals

Patent metadata

Field	Value
Publication number	US-9820475-B2
Application number	US-201214117943-A
Country	US
Kind code	B2
Filing date	May 16, 2012
Priority date	May 16, 2011
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

The disclosure relates to genetically modified swine wherein at least one allele of a SIGLEC1 gene has been inactivated and/or at least one allele of a CD163 gene has been inactivated. Genetically modified swine having both alleles of the SIGLEC1 gene and/or both alleles CD 163 gene inactivated are resistant to porcine reproductive and respiratory syndrome virus (PRRSV). Methods for producing such transgenic swine are also provided.

First claim

What is claimed is:

1. A genetically modified swine comprising inactivating mutations in both alleles of a CD163 gene, wherein the swine does not comprise an inactivating mutation in an allele of a SIGLEC1 gene.

2. A method for producing a genetically modified swine of claim 1 , the method comprising:
enucleating a swine oocyte;
fusing the oocyte with a donor swine somatic cell, the genome of the somatic cell comprising at least one inactivated CD163 allele; and
activating the oocyte to produce an embryo.

3. The method of claim 2 , further comprising transferring the embryo into a reproductive tract of a surrogate swine, wherein the surrogate swine has initiated estrus but has not yet completed ovulation; and wherein gestation and term delivery produces a genetically modified swine whose genome comprises at least one inactivated CD163 allele.

4. The method of claim 3 , further comprising:
mating a female genetically modified swine produced by the method of claim 3 with a male genetically modified swine produced by the method of claim 3 to produce F1 progeny;
screening the F1 progeny to identify genetically modified swine wherein both alleles of the CD163 gene have been inactivated.

5. The method of claim 2 , wherein the at least one allele has been inactivated by partial or complete deletion.

6. The method of claim 2 , wherein the at least one allele has been inactivated using a Cre-lox recombination system.

7. The method of claim 2 wherein the enucleating is performed with a micropipette in a micromanipulation medium.

8. The method of claim 2 wherein the fusing is performed in a fusion medium.

9. The method of claim 2 wherein activating the oocyte comprises incubating the oocyte in the presence of thimerosal.

10. The method of claim 3 wherein the transferring comprises transferring the embryo into an oviduct through an ovarian fimbria of the surrogate.

11. The method of claim 4 , wherein the screening comprises using a polymerase chain reaction (PCR) or Southern Blot.

12. The method of claim 2 , wherein the CD163 allele has been inactivated by replacing exon 7 with scavenger receptor cysteine-rich (SRCR) domain 8 of CD163L.

13. Progeny of a genetically modified swine produced by the method of claim 4 , wherein both alleles of a CD163 gene have been inactivated.

14. The genetically modified swine of claim 1 , wherein the inactivating mutation in at least one of the CD163 alleles results in a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

15. The genetically modified swine of claim 1 , wherein the inactivating mutations in both of the CD163 alleles result in a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

16. The method of claim 4 , wherein the inactivating mutation in at least one-of the CD163 alleles results in a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

17. The progeny of claim 13 , wherein the inactivating mutation in at least one of the CD163 alleles results in a CD163 protein which cannot bind and/or uncoat a porcine reproductive and respiratory syndrome virus (PRRSV).

18. The genetically modified swine of claim 1 , wherein the mutations comprise insertions, deletions, frame shift mutations, nonsense mutations, missense mutations, point mutations, or a combination thereof.

19. The genetically modified swine of claim 1 , wherein the mutations alter the expression or activity of CD163.

20. The method of claim 2 , wherein the somatic cell comprises a fibroblast.

21. The method of claim 2 , further comprising inactivating the CD163 allele by introducing a nuclease into the somatic cell.

22. The method of claim 21 , wherein the nuclease comprises a zinc-finger nuclease or a TAL effector nuclease.

23. The genetically modified swine of claim 1 , wherein the inactivating mutations in the alleles of the CD163 gene comprise partial deletions, complete deletions, or a combination thereof.

24. The genetically modified swine of claim 1 , wherein the inactivating mutations in the alleles of the CD163 gene comprise complete knockouts of the CD163 alleles.

Assignees

• Prather Randall S
• Wells Kevin D
• Whitworth Kristin M
• Univ Missouri

Inventors

• Prather Randall S
• Wells Kevin D
• Whitworth Kristin M

Classifications

• A01K2227/108

  Swine · CPC title

• C07K14/70596

  Molecules with a "CD"-designation not provided for elsewhere · CPC title

• A01K2267/02

  Animal zootechnically ameliorated · CPC title

• A01K2217/15

  Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding · CPC title

• A01K2217/075

  inducing loss of function, i.e. knock out · CPC title